The present invention relates to the field of quantitative microspectroscopy, and in particular to a method for determining the volume of single red blood cells.
Determining the volume of single red blood cells and, based on this measurement, calculating the Mean Cell Volume (xe2x80x9cMCVxe2x80x9d) and the Red Cell Distribution Width (xe2x80x9cRDWxe2x80x9d) is of clinical interest. Usually, systems based on electrical impedance measurement (Coulter Counter) or based on light scattering (Flow Cytometer) are employed (see. e.g., J. B. Henry, xe2x80x9cClinical diagnosis and management by laboratory methodsxe2x80x9d, W. B. Saunders Company, Philadelphia, 1996, pp. 548 ff. or D. H. Tycko, M. H. Metz, E. A. Epstein, A. Grinbaum, xe2x80x9cFlow-cytometric light scattering measurement of red blood cell volume and hemoglobin concentrationxe2x80x9d, Applied Optics 24 (1985), 1355-1365). Impedance counters are complex and expensive instruments that require very careful adjustment and control of instrument and sample parameters. A major disadvantage of flow cytometers is the fact that the parameters of light scattering depend not only on cell volume, but also on cell shape.
In 1983, Gray, Hoffman and Hansen proposed a new optical method for determining the volume of cells in a flow cytometer (M. L. Gray, R. A. Hoffman, W. P. Hansen, xe2x80x9cA new method for cell volume measurement based on volume exclusion of a fluorescent dyexe2x80x9d, Cytometry 3 (1983), 428-432). In this method, the cells are suspended in a fluorescent dye, which is unable to penetrate the cell membrane. The level of fluorescence which is produced when a narrow stream of the cell suspension is excited by a focused laser beam will remain constant until a cell arrives in the illuminated region thereby causing a decrease in fluorescence intensity which is directly proportional to the cell""s volume. In a flow cytometer, a single cell is passing through the laser-illuminated spot within approximately 10 s. Due to this short data acquisition time interval, the electronic detection bandwidth has to be relatively large, which results in a poor signal-to-noise ratio and in a low precision for the volume determination.
The available data acquisition time can be significantly increased by suspending the cells in a stationary sample and applying digital imaging fluorescence microscopy (see P. L. Becker, F. S. Fay, xe2x80x9cCell-volume measurement using the digital imaging fluorescence microscopexe2x80x9d, Biophysical Journal 49 (1986), A465). In the digital fluorescence microscopy approach, a calibration procedure is required in order to determine the cell volume. Recktenwald and co-workers have introduced a method where the calibration is performed by means of optical transparent and non-fluorescent microspheres that are suspended together with the cells (D. Recktenwald, J. Phi-Wilson, B. Verwer, xe2x80x9cFluorescence quantitation using digital microscopyxe2x80x9d, Journal Physical Chemistry 97 (1993), 2868-2870). The volume of individual spheres is determined by measuring their projection area under the microscope and transforming this number into a volume, assuming an ideal spherical shape. The decrease in fluorescence intensity as a result of the spheres"" volume that is being excluded from emitting fluorescence is used as the required calibration parameter. The advantage of this approach is given by the fact that the calibrating particles are located within the sample itself. In other words, a calibration is performed on the very same sample container, and no extra calibration sample is required.
The use of calibration spheres within a cell suspension is not without problems. First, the introduction of the spheres represents an additional step in the workflow. In systems that are designed for high throughput, this additional step would represent a disadvantage. Secondly, Recktenwald and co-workers observed a tendency of the fluorescent dye molecules to settle down on the sphere""s surface, which causes an error. Third, if the optical index of refraction of the spheres does not match well with the liquid""s index, then refraction-based artifacts in the measured fluorescence intensity occur at the edges of the spheres. And, finally, the use of microspheres can represent a problem, if e.g. a thin sample thickness in the order of a few micrometers or less is needed.
In order to overcome the problems in the prior art, it has been suggested (K. W. Berndt, xe2x80x9cMethod for determining the volume of particles suspended in liquidsxe2x80x9d, P-3875, 1997), to design a sample container for the cell suspension that has different optical path lengths in different areas. Preferably, a step-like profile in one of the container windows is employed. In the immediate neighborhood of such step, the change in optical path length is well known, and independent of any possible tilting of the sample container relative to the microscope""s optical Z-axis. The change in intensity due to the well-known step width allows for calibrating the volume determination procedure. The disadvantage of this approach is given by the fact that no cells are allowed at the step, which in practice is not always guaranteed. Also, the production of sample containers equipped with very precise calibration steps in one of the container windows is likely to being costly.
In view of the disadvantages and problems in the prior art as described above, there exists a need for a simple and reliable method for determining the volume of single red blood cells suspended in a liquid.
It is an objective of the present invention to provide a method for determining the volume of single red blood cells or other particles that are suspended in liquids.
According to the present invention, and for the case of red blood cells, the above objective is achieved by depositing a liquid sample that contains suspended red blood cells into an optical cuvette having at least one transparent window, by adding and evenly distributing a fluorescent dye into the liquid that does not leak into the red blood cells, and that is able to absorb excitation light at wavelengths that are only weakly absorbed by the red blood cells, and is able to emit fluorescence light at wavelengths that are only weakly absorbed by the red blood cells; by illuminating the sample at a wavelength that is absorbed by the fluorescent dye, but only weakly absorbed by the red blood cells, by measuring the reemerging fluorescence intensity in an area that contains no red blood cells, by changing the cuvette thickness in that area by a well-defined amount and measuring the reemerging fluorescence intensity in the same area again, by measuring the reemerging fluorescence intensity in an area where a red blood cell resides, by measuring the reemerging fluorescence intensity in an area close to that same red blood cell, and by calculating the volume of the red blood cell based on these fluorescence intensity values and the known change in cuvette thickness.